Administration of leptin

ABSTRACT

A method and composition for administering leptin to a subject. The invention includes suspending isolated native leptin-containing milk fat globules in a suitable medium for administering to a subject. The suspended milk fat globules may be administered orally as well as by intravenous, intramuscular, intraperitoneal, other enteral routes of administration, and other parenteral routes of administration. The invention includes a method for treating growth or maturational-related disorders in newborns as well as subjects having conditions that can be treated by the administration of leptin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation application of U.S. application Ser. No.09/302,117 filed Apr. 29, 1999, now U.S. Pat. No. 6,475,984, of whichbenefit and priority is claimed hereby and the entirety of which areincorporated by reference.

BACKGROUND

Leptin, the protein product of the ob gene, is an important circulatingsignal for regulating body weight, food intake, and energy metabolism inmammals (Zhang Y et al., Nature 372: 425-432, 1994). These actions areelicited through the binding of leptin to a high affinity receptor inthe hypothalamus (Tartaglia LA et al., Cell 83: 1263-1271, 1995).Although leptin Was initially described as a satiety factor thatregulates the size of adipose tissue, leptin has many other diversebiological functions. These functions are elicited by the binding ofleptin to receptor proteins that are expressed in numerous tissues.

Leptin has been shown to correct the sterility defect inleptin-deficient mice and accelerate puberty when administered to normalmice (Chehab et al. Nature Genet. 12:318-320, 1996). Humans defective ineither leptin or the leptin receptor are sterile and sexually immature,supporting leptin's role in reproduction (Stroebel et al., Nature Genet.18: 213-215, 1998; Clement et al., Nature 392: 398-401, 1998). Otherroles for leptin include a regulator of hematopoeisis (Cioffi et al.,Nature Medicine 2: 585-589, 1996; Gainsford et al., Proc. Natl. Acad.Sci. USA 93. 14564-14568, 1996), angiogenesis (Bouloumie et al., Circ.Res. 83: 1059-1066, 1998; Sierra-Honigmann et al., Science 281:1683-1686, 1998), glucose metabolism (Kamohara et al., Nature 389:374-377, 1997), and proinflammatory immune responses (Loffreda et al.,FASEB J 12:57-65, 1998; Lord et al., Nature 394: 897-901, 1998). Sincemalnutrition is the leading cause of diminished immunity and increasedsusceptibility to infection, leptin therapy may also augment the immuneresponse in compromised individuals (Flier, Nature Medicine 4:1124-1125, 1998).

Although first thought to be produced exclusively by the adipocyte (fatcell), it is now known that leptin is produced in the placenta (Hassinket al., Pediatrics 100:e1-e6, 1997; Masuzaki et al., Nature Med. 3:1029-1033, 1997), gastric epithelium (Bado et al., Nature 394:790-793,1998), and the mammary gland, as more fully described below(Smith-Kirwin et al., J. Clin. Endocrinol. Metab. 83: 1810-1813, 1998,herein incorporated by reference). The function of placental leptinseems to be as a regulator of fetal growth. Prematurity accounts for alarge proportion of infant morbidity, primarily due to respiratorydistress, immaturity of organ systems, and poor nutrition. As more fullydescribed below, the premature infant is leptin-deficient, due to earlyseparation from the placenta at a time when they have inadequate adiposetissue. A method to administer leptin to premature and/or poorly growinginfants is highly desirable.

Previous leptin therapies rely on the use of recombinant leptin. Theadministration of recombinant leptin is performed intravenously,intramuscularly, intraperitoneal, and through other parenteral routes totreat obesity, diabetes, and reproductive abnormalities. The amount ofleptin that can be delivered by these means is limited by recombinantleptin's poor solubility and by local reactions in skin that occur inresponse to high doses (Friedman and Halaas, Nature 395: 763-770, 1998).In rodent models, it has been shown that the ability to optimize themeans of administration of leptin may greatly influence its therapeuticeffectiveness. Leptin has been shown to be more potent when administeredas a subcutaneous infusion than when administered by dailyintraperitoneal injections (Halaas et al., Proc. Natl. Acad. Sci. USA94: 8878-8883, 1997). Direct administration of leptin into thecerebrospinal fluid (Halaas et al., Proc. Natl. Acad. Sci. USA 94:8878-8883, 1997) and by gene therapy means (Chen et al., Proc. Natl.Acad. Sci. USA 93: 14795-14799, 1996; Murphy et al., Proc. Natl. Acad.Sci. USA 94: 13921-13926, 1997) have also been shown to be effective forweight loss in rodents, but for safety and ethical concerns have notbeen tried in humans.

Previous methods involving leptin therapies rely on the use ofrecombinant leptin. Purification of recombinant leptin involvesdenaturation of the protein and subsequent renaturation steps. Therenatured leptin must then be solubilized, which is problematic becausethe recombinant leptin aggregates at high concentrations and muchprotein loss occurs (Guisez et al., Protein Expression Purification 12:249-258, 1998). The renatured leptin must then be tested forbloactivity.

Therefore, it is desirable to provide a method for administering leptinthat does not require the use or purification of recombinant leptin thatcan be administered orally, intraveneously, subcutaneously,intramuscularly, intraperitoneally or by other parenteral means. Asstated above, a method to administer leptin to premature and/or poorlygrowing infants is highly desirable.

As more fully described below, the present invention overcomes theproblems associated with previous forms of leptin therapy and includes anovel method and composition for administering native leptin that can beadministered to subjects who have deficient leptin levels or requireleptin therapy to treat a disorder.

SUMMARY OF THE INVENTION

The present invention includes a method and composition of native leptinin milk fat globules. This composition may be administered orally,intravenously, intramuscularly, intraperitoneally, or by other enteralor other parenteral means. In one embodiment, the invention includes amethod for treating growth-related problems in newborns.

The invention includes a composition for the administration of leptincomprising isolated milk fat globules wherein the milk fat globulescontain a native form of leptin.

The invention further includes isolated milk fat globules that arederived from a milk-producing mammal that produces milk fat globulescontaining a native form of leptin. The mammal may be selected from thegroup consisting of humans, cows, rats, mice, and goats.

The invention includes a medium that is effective for suspending themilk fat globules and is safe to administer to humans. Preferably themedium is selected from the group consisting of physiologic saline,water, intravenous fluid, milk, human breast milk, baby formula, and anyother medium that is safe for human or infant administration.

The invention includes suspending the isolated milk fat globules in themedium at a concentration between about 50 ng/ml leptin and about 100ng/ml leptin. Preferably, the isolated milk fat globules are suspendedat a concentration of about 75 ng/ml.

Further, the invention includes isolated milk fat globules where atleast a portion of the leptin is contained within the milk fat globules.

The invention includes a method for administering leptin that includesadministering to a subject an effective amount of isolated nativeleptin-containing milk fat globules where the isolated nativeleptin-containing milk fat globules have been suspended in a medium toproduce suspended milk fat globules.

The invention includes subjects that have a condition selected from thegroup consisting of obesity, sterility, sexual immaturity, malnutrition,compromised immunity, psychiatric disorders, diabetes, and subjects witha condition that can be treated by the administration of leptin.

Still further, the invention includes a subject selected from a groupconsisting of a premature infant, a full-term infant having low leptinlevels, a low birth weight infant, and an infant with a condition thatcan be treated with leptin.

The present invention further includes administering an effective amountof suspended milk fat globules by an administration method selected fromthe group consisting of oral administration, subcutaneousadministration, intramuscular administration, intravenousadministration, intraperitoneal administration, and other enteral orparenteral routes of administration.

Still further, the present invention includes a method for administeringleptin to infants that includes administering to the infant an effectiveamount of isolated native leptin-containing milk fat globules whereinthe isolated native leptin-containing milk fat globules are suspended ina medium to produce suspended milk fat globules.

The invention includes a medium that is effective for suspending themilk fat globules and is safe to administer to infants. Preferably, themedium is selected from the group consisting of physiologic saline,water, intravenous fluid, milk, human breast milk, baby formula, soymilk, and any other medium that is safe for infant administration.

The invention includes administering an effective amount of leptin to aninfant. Preferably, the effective amount is about 0.5 ng to about 1.0 ngof leptin per gram of infant body weight per feeding.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a plot of leptin concentration (ng/ml) as a function of timeafter the addition of pancreatic lipase.

FIG. 2 is a plot of the average mid arm/head circumference ratio forbreast-fed infants (leptin) and formula-fed infants (no leptin) as afunction of time (weeks).

FIG. 3 is a RT/PCR analysis of leptin mRNA in mammary gland and adiposetissue. Total RNA was reversed transcribed from either mammary gland(lanes 1,3,5) or adipose (lanes 2,4,6) tissue and amplified with PCRprimers to either leptin (lanes 1-2), both leptin and β-actin (lanes3-4) or β-casein (lanes 5-6). Markers (M) are 1000, 700, 525, 500, 400,300 bp. The size of the leptin RT/PCR product is 348 bp; β-actin is 592bp; β-casein is 329 bp.

FIG. 4 shows immunohistochemical detection of leptin in human breasttissue and mammary epithelial cells. Sections were stained for leptin orepithelial membrane antigen as described in the Methods. Colorimetricdetection was with DAB (brownish purple stain) for panels A, D, and F orAEC (red stain) for panels B, C, and E. (A) Human breast tissue stainedfor leptin, counterstained with methyl green; (B) Cultured HMEC onMatrigel, no primary antibody; (C) Cultured HMEC on Matrigel stainedwith leptin; (D, E) Epithelial cells from cytospins of 6 mo post-partumbreast milk stained with either (D) leptin, counterstained with methylgreen or (E) epithelial membrane antigen, counterstained with Mayer'shematoxylin; (F) milk fat globules from cream of breast milk stainedwith leptin. (A-E), 245× magnification; (F), 612× magnification.

FIG. 5 is a plot showing the effect of sonication on leptin levels in(A) whole and (B) skim milk. Milk samples were prepared and analyzed forleptin levels by radioimmunoassay as described in the Methods. (▪)sonicated; (□) not sonicated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on studies that show leptin is producedby the mammary gland and packaged in milk fat globules (MFGs). Theinclusion of leptin in MFGs is believed to confer protection of leptinby proteases and the acid environment of the stomach. It may alsoprovide a means by which leptin is solubilized as leptin is believed tobe bound to other proteins in the circulation which modulate itsbioactivity and bioavailability (Houseknecht et al., Diabetes 45:1638-1643, 1996; Hill et al., Int. J. Obesity 22: 765-770, 1998).Purified recombinant leptin is insoluble and must be denatured beforesolubilization. Both Guisez et al. (Protein Expression Purification 12:249-258, 1998) and Campbell et al. (Biochem. Biophys. Res. Comm. 247:654-658, 1998) provide evidence for the binding of leptin to fattyacids, in particular oleic acid, palmitic acid, and arachidonic acid.These fatty acids are the same fatty acids that comprise the core of theMFG.

Purified MFGs provide a solubilized, stable, and native leptinpreparation. Evidence for leptin inside the globule is derived fromseveral recent studies, which are described in Smith-Kirwin et al. (J.Clin. Endocrinol. Meta. 83: 1810-1813, 1998) herein specificallyincorporated by reference and described in Example 1 below. Brieflysummarized, the following points support the conclusion that leptin isassociated with MFGs. First, increased leptin levels are found in thecream portion of whole milk and in isolated milk fat globules. Second,MFGs are positive for leptin by immunohistochemical staining, as arebreast tissue, cultured mammary epithelial cells, and secretoryepithelial cells present in human milk. Finally, MFGs must be disruptedby either mechanical or enzymatic means to detect leptin byradioimmunoassay. This suggests that the epitopes recognized by theleptin antibody are normally not accessible in the intact MFG.

Low levels of immunodetectable leptin are found in whole milk. However,when whole milk is homogenized by mechanical disruption, leptin levelsincrease 100-fold. These results indicate that leptin is bound to asubstance in the milk fat. This substance is the MFG.

With reference now to FIG. 1 and more fully explained in Example 2,isolated MFGs were treated with pancreatic lipase to determine thelevels of leptin in the MFGs. FIG. 1 shows treatment of whole milksamples with 50 U/ml pancreatic lipase, which acts to breakdowntriglyceride in the MFG, and measurement of released, immunodetectableleptin levels over time. Leptin release occurs in two phases, an initialrapid release which likely represents a loose association of leptin withthe MFG, and a slower release, which indicates leptin is being releasedfrom a bound source, perhaps the fatty acids in the MFG.

A difficulty with the oral administration of leptin is that leptin isacid-labile and would not survive the acidic environment of thedigestive system. The packaging of leptin in the MFG provides an oralmeans of administering leptin systematically. The MFG consists of atriglyceride core surrounded by a membrane that is pinched off from theapical membrane of breast epithelial cells and envelopes the milk fat.

MFGs can be isolated from any milk-producing mammal that producesleptin-containing MFGs in their milk. These mammals include but are notlimited to humans, cows, rats, mice, and goats. As used herein “isolatedMFGs” refers to native leptin-containing MFGs that have been isolatedfrom any milk-producing mammal that produces native leptin-containingMFGs. Several methods to isolate MFGs have been described and are wellknown in the art such as the method described in Giuffrida et al., J.Prot. Chem. 17: 143-148, 1998 herein specifically incorporated byreference and Patton and Huston, Lipids 21: 170-174, 1986 hereinspecifically incorporated by reference. A preferred method is thetechnique described by Patton and Huston, in which MFGs were isolatedfrom cow, goat, and human milk by centrifuging the MFGs through anoverlaying buffer layer. It has been shown that 85% of the xanthineoxidase and alkaline phosphatase activities are removed from MFGs byfour successive water washes (Zittle et al., J. Dairy Sci. 39: 528-535,1956 herein specifically incorporated by reference). Since a significantproportion of leptin is loosely bound to the MFG as described above, themethod described by Patton and Huston would be expected to minimize theloss of leptin from MFGs.

The leptin concentration in MFGs may be determined by either sonicationor lipase treatment of the MFGs followed by analysis of the treated milksample by radioimmunoassay for immunodetectable leptin. Prior toadministering the isolated MFGs to a subject, the isolated MFGs shouldbe resuspended in a medium to produce suspended MFGs. As used herein,“suspended MFGs” means a solution in which the isolated MFGs have beenreduced in concentration or diluted by the medium. The MFGs may beresuspended in a medium including, but not limited to, physiologicsaline (0.9% wt/vol NaCl), water, intravenous solutions, human breastmilk, milk, baby formula, or any other medium that is safe for humanadministration. The isolated MFGs are resuspended at a concentrationbetween about 50 ng/ml leptin to about 100 ng/ml leptin and preferablyresuspended at a concentration of 75 ng/ml.

The MFGs should be resuspended in a medium that is appropriate for thetype of administration. If the MFGs are being administered orally, theMFGs should be resuspended in a medium appropriate for consumption. Anappropriate medium includes, but is not limited to, human breast milk,milk, baby formula, soy milk, water, or other liquid safe for humanconsumption. If the MFGs are to be injected intravenously,subcutaneously, intraperitoneally, or intramuscularly, the MFGs shouldbe suspended in an appropriate medium for injection including, but notlimited to, physiologic saline, water, and other intravenous solutions.Additional supplements including, but not limited to, nutrients, drugs,or other substances may be added to the suspended MFGs prior to, during,or along with the administration of an effective amount of the suspendedMFGs to a subject provided that the addition of the additionalsupplement does not prevent the effective administration of leptin tothe subject.

An effective amount of suspended MFGs may be administered by any of theabove referenced methods to subjects requiring leptin therapy to treat adisorder, also referred to herein as a “condition.” These conditionsinclude, but are not limited to, obesity, sterility, sexual immaturity,malnutrition, compromised immunity, psychiatric disorders, diabetes, andsubjects with a disorder that would can be treated by the administrationof leptin. Further, leptin may be administered to a subject to assist inthe development of the brain.

Of particular importance is the applicability of the present inventionto the treatment of infants. The function of placental leptin seems tobe as a regulator of fetal growth. Leptin levels in cord blood arehighly correlated with both the infant's birth weight and body massindex (Koistinen et al., J. Clin. Endocrinol. Metab. 82: 3328-3330,1997; Harigaya et al., J. Clin. Endocrinol. Metab. 82: 3281-3284, 1997;Schubring et al., J. Clin. Endo. Metab. 82: 1480-1483, 1997; Matsuda etal., J. Clin. Endo. Metab. 82: 1642-1644, 1997; Tamura et al., Obstet.Gynecol. 91: 389-395, 1998; Marchini et al., Pediatrics 101: 429-432,1998). Both large-for-gestational age and infants of diabetic mothershave higher cord blood leptin concentrations thanappropriate-for-gestational age infants (Lepercq et al., Diabetes 47:847-850, 1998). Premature infants have low leptin levels, which increase3-fold in mothers who receive steroids antenatally (Shekhawat et al.,Pediatr. Res. 43: 3338-3343, 1998). Interestingly, within 48 hours ofdelivery, the concentration of leptin in large-for-gestational age andappropriate-for-gestational age babies decreases to the level insmall-for-gestational age infants. These low leptin levels continue to 7days of age. Helland et al. (Pediatrics 101: e12, 1998) have shown thatleptin levels also decrease from birth to 4 weeks of age, but moderatelyincrease thereafter. Furthermore, at all time points examined, leptinlevels are significantly higher in female infants as compared to males.

As mentioned earlier, prematurity accounts for a large proportion ofinfant morbidity, primarily due to respiratory distress, immaturity oforgan systems, and poor nutrition. The premature infant isleptin-deficient, due to early separation from the placenta at a timewhen they have inadequate adipose tissue stores.

Leptin concentrations were measured at semi-weekly intervals from birththrough discharge (range 1-77 days) in 11 premature infants requiringmechanical ventilation (gestational age=28±2 weeks, birth weigh=986±189g, mean±S.D.). At the time of leptin sampling, anthropometricmeasurements were obtained, and calculations of mid/armcircumference/head circumference ratio, and upper arm fat were made. Onehundred fifty six measurements of leptin concentrations were obtained inthis patient population (mean±SD=1.24±0.63 ng/ml, range=0-3). Using amultiple linear regression model, the best variables in this samplepopulation to predict leptin levels were determined. The values thatbest predicted leptin, in descending order of variance, were: midarm/head circumference ratio, lower arm circumference, calfcircumference, postnatal age, upper arm fat, mid thigh circumference,daily weight, birth weight, and abdominal girth (r value=0.71,r-squared=0.51, p<0.00001).

A multiple linear regression using the variables measured in theinvestigation and the ratio of mid arm/head circumference as thedependent variable to reflect adequacy of growth in the premature infantwas performed (Spear et al., Pediatr. Res., 45(4):291A, 1999 hereinspecifically incorporated by reference). It is well known in the artthat Georgieff et al. (J. Pediatr. 109: 316-321, 1986), hereinspecifically incorporated by reference, determined that using the midarm/head circumference ratio is a better measurement of growth ininfants than using the infant's birth weight. In this model, the bestpredictors of the mid arm/head circumference ratio, in descending orderof variance, were: the mid thigh circumference, leptin,post-conceptional age, triceps skin fold, upper arm circumference,intake, calf circumference, crown rump length, and total body length(r=0.9, r-squared=0.81, p<0.00001). Of most importance, leptin itselfaccounted for 27% of the variance in mid arm/head circumference ratio.Leptin values in this group of premature infants were both affected byand predictive of neonatal growth. This expands the potential role ofleptin as both an intrauterine and neonatal growth factor.

For the newborn infant (both full-term and premature), the physicalbenefits of breast-feeding are multiple and provide such diverseattributes as protection from infections (upper respiratory, intestinaland middle ear), and a decrease in atopic diseases. Many components ofhuman breast milk have also been shown to be necessary for developmentof the brain, intestinal tract, spinal cord, and retina (Crawford MA etal., A. J. Clin. Nutr. 31:2181-2185, 1978). In general, thebioavailability of human milk components is remarkably high and issuperior compared to cow's milk or formula (Fuchs, A R: Physiology andEndocrinology of Lactation p. 549-577. In Obstetrics: Normal and ProblemPregnancies, 1986).

With reference now to FIG. 2, in a longitudinal study comparing variousgrowth parameters in formula and breast-fed infants, breast-fed infantswere found to grow at a faster rate than formula-fed infants. Leptinlevels in breast milk increased during the 12 weeks of the study. Inearly and transitional milk, leptin levels were ˜20 ng/ml; in maturemilk, leptin levels were ˜70 ng/ml, similar to that observed below inExample 1 regarding leptin levels in established breast milk(73.22±39.03 ng/ml, n=8). Together, the studies in premature andfull-term infants indicate that leptin acts as a growth factor and notas a satiety factor in infants.

The present invention may be used to administer leptin to prematureinfants, low-birth weight infants, full-term infants with low leptinlevels, and infants having a condition that may be treated with theadministration of leptin. As used herein “infant” refers to normalinfants, premature infants, low-birth weight infants, full-term infantswith low leptin levels, and infants having a condition that may betreated with the administration of leptin. Prior to administering theisolated MFGs to an infant, the isolated MFGs should be resuspended in aliquid to produce suspended MFGs. The MFGs are preferrably resuspendedin a medium including, but not limited to, physiologic saline (0.9%wt/vol NaCl), water, intravenous solutions, human breast milk, milk,baby formula, or any other medium that is safe for infantadministration. The isolated MFGs are preferrably resuspended at aconcentration between about 50 ng/ml leptin to about 100 ng/ml leptinand most preferably resuspended at a concentration of 75 ng/ml.

The MFGs should be resuspended in a medium that is appropriate for thetype of administration. If the MFGs are being administered orally or byother enteral means, resuspending the MFGs in a medium appropriate forinfant consumption including, but not limited to, human breast milk,milk, baby formula, soy milk, water, or other liquid safe for humanconsumption is preferred. The isolated MFGs may be suspended in babyformula to provide a formula-feeding baby with a source of leptin.Further, the isolated MFGs may be added to human breast milk even if theinfant is currently feeding from breast milk to supplement the breastmilk with additional leptin.

If the MFGs are to be injected intravenously, subcutaneously,intraperitoneally, or intramuscularly, the MFGs should be suspended inan appropriate medium for injection to an infant. An appropriate mediumincludes, but is not limited to, physiologic saline, water, andintravenous solutions.

Additional supplements including nutrients, drugs, or other substancesmay be added to the suspended MFGs prior to administering the MFGs tothe infant provided that the addition of the additional supplement doesnot prevent the effective administration of leptin to the infant. Aneffective amount of suspended MFGs is administered to the infant. MFGsare administered to infants at a dosage between about 0.5 ng leptin pergram body weight per feeding and about 1.0 ng leptin per gram bodyweight per feeding.

EXAMPLE 1 Determination of Leptin Levels in Breast Milk

Measurement of leptin in breast milk: All breast milk was obtained fromdonors who voluntarily consented to participate in the study. They werehealthy women from the Research Department, between 25-35 yr of age, whoplanned to breast-feed and had delivered healthy full term infants. Milkwas collected by either hand expression or a hand-held electric pump andstored frozen at −20° C. For leptin analysis, milk samples were thawedovernight in the refrigerator. Skim milk was prepared by centrifugationof whole milk at 13000 rpm for 10 min at 4° C. to separate milk fat fromthe liquid phase. A spatula was used to remove the layer of fat. Milksamples were sonicated for 3-10 sec bursts with 20 sec cooling intervalsusing a Branson Model W180 sonicator with a stepped microtip.Radioimmunoassay (Linco Research, St. Charles, Mo.) for serum leptin wasperformed as described by the manufacturer.

Cvtospin cell preparations: Human mammary epithelial cells were preparedfor cytospin from at least 20 ml breast milk. Milk was spun at 2500 rpm,and the liquid and fat layers were removed. The pelleted cells wererinsed once in HBSS and then resuspended in HBSS. The cell suspensionwas transferred to disposable cytofunnels with attached cytospin slidesand spun at 1000 rpm for 10 min in a Cytospin 3 (Shandon Inc.,Pittsburgh, Pa.). Slides were stored frozen at −20° C.

Cell Culture: Human Mammary Epithelial Cells (HMEC) were obtained fromClonetics (San Diego, Calif.) and grown in serum-free MEGM medium(Clonetics). HMEC cells were mixed 1:1 with cold undiluted Matrigel(Collaborative Research, MA) by methods known to one skilled in the art,such as the method described in Gomm et al. (J. Cell Physiol. 171:11-19)herein specifically incorporated by reference. Twenty-four well plateswere seeded with this cellular mix and warmed to 37° C. for at least 30min before being overlaid with the growth medium. Cells were allowed togrow for up to 3 weeks in culture during which time they continued todivide and fuse to form large aggregates of cells from which ducts werecommonly seen.

Immunohistochemistry: Normal frozen breast tissue was obtained fromChristiana Care, Inc. (Christiana, Del.), and 10 μm sections were cutand placed onto glass slides. These sections and the cytospin cellsamples were fixed in Streck Tissue Fixative (Streck Laboratories Inc.,Omaha, Nebr.) for 5 min at room temperature. HMEC cells grown inMatrigel were fixed overnight at 4° C. in the same fixative before thecellular gel was removed, mounted in 10% Tragacanth gum, snap-frozen inisopentane, and 10 μm sections prepared. Samples were then blocked withPeroxo-block and CAS block (Zymed, San Francisco, Calif.) according tothe manufacturer's instructions. Superblock (Research Genetics,Huntsville, Ala.) was utilized to block background staining fromMatrigel proteins. Polyclonal rabbit anti-leptin (RDI, Flanders, N.J.)was diluted 1:100 with serum blocking buffer and incubated for 60 min atroom temperature. Antibody to epithelial membrane antigen (Zymed) wasused neat; milk fat globule membrane antibody (Novocastra Laboratories,Ltd., Burlingame, Calif.) was diluted 1:50. A kit obtained from Zymedwas used for subsequent steps for the biotinylated labeled secondaryantibody and calorimetric detection with either DAB or AEC chromagen.

RT-PCR: Human mammary gland total RNA was obtained from Clontech (PaloAlto, Calif.), and adipose RNA was from Invitrogen (Carlsbad, Calif.).One microgram of total RNA was reverse-transcribed using avianmyeloblastosis virus and oligo (dT) primer (Promega, Madison, Wis.),according to the manufacturer's instructions. Amplification of the cDNAsequence was performed by methods well known to one skilled in the art.For assessment of the relative levels of leptin to β-actin, a multiplexRT/PCR approach known to those skilled in the art was used, such as themethod described by Dukas et al. (Anal. Biochem. 215:66-72, 1993) hereinspecifically incorporated by reference and Hassink et al. (Pediatrics100:e1-e6, 1997) herein specifically incorporated by reference. Primersequences for leptin and β-actin were those known in the art anddescribed previously in Hassink et al. (Pediatrics 100:e1-e6, 1997)herein specifically incorporated by reference. β-casein primers were:

CASF1: 5′ ATTCTGCCTCTTGCTCAGCCTGC3′ (SEQ ID NO: 1); and

CASR1: 5′ AGCTCTCTGAGGGTAGGGCACCAC3′ (SEQ ID NO: 2).

PCR products were separated on a 4% NuSieve (FMC Bioproducts, Rockland,Me.) agarose gel and stained with ethidium bromide.

RT/PCR analysis of total RNA from mammary gland revealed the presence ofleptin mRNA at a level comparable to that of adipose tissue (FIG. 3).Sequencing of the leptin cDNA product revealed complete sequenceidentity to that of placental and adipose leptin (GenBank accessionnumber D9487). FIG. 3 also shows expression of β-casein, a marker ofmammary epithelial cell differentiation, in the mammary gland, but notthe adipose tissue RNA.

Immunohistochemical staining with a polyclonal antibody to human leptinshowed leptin production by ductal epithelial cells of human breasttissue (FIG. 4A). FIG. 4C shows that cultured human mammary epithelialcells maintained on a Matrigel substrate also produce leptin. Finally,leptin was shown to be present in the secretory epithelial cells ofbreast milk (FIG. 4D). These cells, which are the predominant cell typeshed into milk after the second month of lactation, were identified asepithelial cells by their positive staining for epithelial membraneantigen (FIG. 4E). Other abundant cell types in human milk includeneutrophils and macrophages; these cell types did not express leptin.Interestingly, intense immunostaining for leptin was observed innon-cellular vesicles of human milk (FIG. 4F). These vesicles alsostained positively for milk fat globule protein (not shown). Theseresults indicate that leptin is either present in, or associated with,the milk fat globules of breast milk.

Immunoreactive leptin levels were higher in whole milk as compared toskim milk (FIG. 5A). However, it was critical to sonicate the milksample to detect the high levels of leptin in whole milk. Sonication ofskim milk samples made no difference in immunodetection of leptin (FIG.5B). These data, together with the immunolocalization of leptin to milkfat globules described above, suggest that leptin is sequestered withinthese vesicles and not detectable by radioimmunoassay. Sonicationdisrupts the membrane vesicles, and allows subsequent immunodetection ofleptin.

This example shows that human mammary epithelial cells produce andsecrete leptin. Furthermore, leptin is associated with milk fatglobules, which partition into the lipid fraction of whole milk. Theseresults explain why leptin is found in higher concentration in whole ascompared to skim milk.

Without intending to be bound by theory, it is believed that milk fatglobules are derived from the apical plasma membrane of the epithelialcell and the secretory vesicle membrane of the Golgi apparatus. Theglobules therefore consist of a lipid core enclosed by membrane andmembrane-bound proteins that are produced in the Golgi. Leptin becomespart of the milk fat globule as it is processed in the Golgi apparatus.It is likely that the association of leptin with these globules confersa protective effect against degradation of leptin by the infantdigestive tract.

Surprisingly, average leptin concentrations in breast milk (73.22±39.03ng/ml, mean±SE, n=8) were higher than serum leptin levels in normal(7.5±9.3 ng/ml), obese (31.3±24.1 ng/ml), pregnant (29.8±17.0 ng/ml), ornursing (8.93±0.96 ng/ml) individuals. Although sonication was necessaryto detect the high levels of leptin in whole breast milk, sonication ofskim milk did not increase leptin levels. Thus, the mammary glandappears to produce high quantities of leptin, in agreement with theRT/PCR data (FIG. 3) showing similar levels of leptin mRNA in adiposeand mammary gland tissues. Sequencing of the leptin cDNA productrevealed complete sequence identity to that of placental and adiposeleptin.

The low level of leptin that is detected in skim milk may arise from thematernal circulation and not the mammary gland. This would account forthe high correlation between leptin levels in maternal serum and skimmilk. It also would explain why sonication did not increase leptinlevels in skim milk. However, the predominant source of leptin in breastmilk appears to be the mammary gland. Putative roles of leptin in breastmilk include a neonatal growth factor and a regulator of neonatal foodintake.

EXAMPLE 2 Determination of Leptin Content in Lipase-Treated Milk Samples

In Example 1 it was critical to sonicate the milk sample to detect thehigh levels of leptin in whole milk. Occasionally, with high fat milksamples, sonication interfered with the leptin radioimmunoassay, and nopellet was formed in the assay. Without intending to be bound by theory,this is probably due to the formation of micelles in high fat sonicatedmilk samples. To attempt to circumvent this problem, we used pancreaticlipase to disrupt the MFG as described below.

Six hundred microliters of whole milk was incubated in a 37° C. waterbath for 10 min. Six microliters of a 1 M sodium bicarbonate solutionwas then added in order to adjust the pH of the sample to approximately8.0, the optimal pH for pancreatic lipase activity. Two and four-tenthsmicroliters (30 U) of pancreatic lipase (Sigma Chemical Co., St. Louis,Mo.) was added to the milk sample, and the sample was incubated withconstant shaking at 37° C. for 60 min. The sample was placed on ice andthen assayed for leptin levels by radioimmunoassay as described above.The data in Table 1 show that it is possible to detect leptin levels inlipase-treated milk, even when the milk sample has a high fat content.

TABLE 1 Leptin levels (ng/ml) in sonicated and lipase-treated milksamples Whole Whole-Sonicated Whole-Lipase-treated 0.68 No pellet 6.49

EXAMPLE 3 Isolation of Milk Fat Globules and Determination of LeptinLevels

MFGs were isolated by methods known to those skilled in the art such asthe method described in Giuffrida et al. (J. Prot Chem 17:143-148, 1998)herein specifically incorporated by reference. Fresh milk was collectedfrom mothers enrolled in a Newborn growth study. Milk (10 ml) wastransferred to a 15 ml centrifuge tube and spun at 2000 g for 20 minutesat room temperature. The supernatant (cream) was collected by pipettingand transferred to a 1.5 ml microfuge tube and centrifuged at 6° C. for70 minutes at 100 g. MFGs were then collected by ‘scooping’ out thecongealed cream with a sterile spatula, and transferred to a new 1.5 mlmicrofuge tube. MFGs were washed 3× with 0.9% (wt/vol) NaCl andcentrifuged at 2000 g for 30 min at 6° C. Finally, MFGs were resuspendedin a volume of saline commensurate with the starting volume of milk, inthis case 10 ml. Leptin levels were measured by radioimmunoassay byusing a kit commercially available from Linco, according to themanufacturer's instructions. Leptin levels in isolated intact MFGs were<0.5 ng/ml. Treatment of the MFGs with 50 U/ml pancreatic lipase for 60min resulted in a final leptin concentration of 90.92 ng/ml.

To determine whether leptin could be fully recovered from milk that hadbeen frozen and thawed, the same procedure was followed as describedabove. Leptin levels were compared in intact MFGs (untreated), sonicatedMFGs, and MFGs treated with lipase. The data in Table 2 indicate thatthe recovery of leptin in MFGs from thawed milk is not as good as freshmilk. Thus, the preferred method of isolating MFGs is from fresh milk.

TABLE 2 Leptin levels (ng/ml) in MFGs purified from milk samples MFGsMFGs MFGs Sample No. untreated Sonicated lipase-treated 1 <0.5 <0.5 0.562 <0.5 <0.5 1.65 3 49.4 68.03 68.03 4 <0.5 <0.5 0.5 5 <0.5 1.04 <0.5 6<0.5 <0.5 1 7 <0.5 <0.5 0.7 8 <0.5 <0.5 1.65

Those persons skilled in the art will therefore readily understand thatthe present invention is susceptible of a broad utility and application.Many embodiments and adaptations of the present invention other thanthose herein described, as well as many variations, modifications andequivalent arrangements, will be apparent from or reasonably suggestedby the present invention and the foregoing description thereof, withoutdeparting from the substance or scope of the present invention.Accordingly, while the present invention has been described herein indetail in relation to its preferred embodiment, it is to be understoodthat this disclosure is only illustrative and exemplary of the presentinvention and is made merely for purposes of providing a full andenabling disclosure of the invention. The foregoing disclosure is notintended or to be construed to limit the present invention or otherwiseto exclude any such other embodiments, adaptations, variations,modifications and equivalent arrangements, the present invention beinglimited only by the claims appended hereto and the equivalents thereof.

1. A method for stimulating growth in an infant, said method comprisingthe steps of: administering isolated and substantially purified milk fatglobules containing an effective dosage of leptin to the infant, andstimulating growth in the infant.
 2. The method of claim 1, wherein theeffective dosage of leptin is in the range of about 0.5 ng to about 1.0ng of leptin per gram of infant body weight.
 3. The method of claim 1,wherein said administering step is performed orally.
 4. The method ofclaim 1, wherein said administering step is performed by anadministration route selected from the group consisting ofintravenously, subcutaneously, intraperitonally, and intramuscularly. 5.The method of claim 1, wherein the isolated and substantially purifiedmilk fat globules containing the effective dosage of leptin aresuspended in a medium appropriate for administration to an infant at aconcentration in the range of about 50 ng/ml leptin to about 100 ng/mlleptin.
 6. The method of claim 5, wherein the medium is selected fromthe group consisting of physiologic saline, breast milk, milk, water,and baby formula.
 7. The method of claim 6, wherein the medium is babyformula.
 8. The method of claim 5, wherein the medium is administered byan administration route selected from the group consisting ofintravenously, subcutaneously, intraperitonally, and intramuscularly. 9.The method of claim 1, wherein the infant is a formula-fed infant. 10.The method of claim 1, wherein the infant is a small-for-gestational ageinfant.
 11. The method of claim 1, wherein the infant is a prematureinfant.
 12. The method of claim 1, wherein the infant has low leptinlevels.